Cryoconserved Mature Dendritic Cells

ABSTRACT

The invention relates to a method for producing ready to use, antigen loaded or unloaded, cryoconserved mature dendritic cells especially for the production of a vaccine containing said dendritic cells, wherein immature dendritic cells are cultivated in the presence of suitable maturation stimuli and the mature dendritic cells thus obtained are frozen. The dendritic cells can be loaded with antigen before freezing. The invention also relates to a vaccine which can be obtained according to the inventive method and to a composition containing frozen, mature dendritic cells which are loaded with antigen.

TECHNICAL FIELD

The present invention relates to a method for the preparation ofready-for-use cryoconserved mature dendritic cells, especially for thepreparation of a vaccine which contains such dendritic cells, whereinimmature dendritic cells are cultured in the presence of suitablematuring stimulants, and the mature dendritic cells thus obtained arefrozen. Prior to or after freezing, the dendritic cells may be loadedwith antigen. The invention also relates to a vaccine obtainable by themethod according to the invention, and to a composition containingfrozen mature antigen-loaded dendritic cells.

BACKGROUND OF THE INVENTION

Dendritic cells (hereinafter briefly referred to as “DC”) areantigen-presenting cells which influence the immune system byinteracting with lymphocytes. Most DCs exhibit an immunostimulatoryactivity. These classical DCs can induce the formation of helper andkiller T cells in vivo in different ways (“nature's adjuvant”). Inparticular, immature DCs which occur in peripheral tissues have thecapability of binding antigens and preparing immunogenic MHC peptidecomplexes therefrom (“antigen processing mode”). Upon the action ofmaturation-inducing stimulants, such as inflammatory cytokines, theseimmature DCs develop into potent T-cell stimulants through an increasedformation of adhesion and costimulatory molecules (“T-cell stimulatorymode”). At the same time, the cells migrate into secondary lymphaticorgans to select and stimulate rare antigen-specific T cells. It couldbe shown that DCs which were isolated from tissues or blood and loadedwith antigen in vitro were immunogenic after back injection as matureDCs in vivo.

Recently, it could be shown that DCs can induce CD4+ and CD8+ T-cellimmunity in both healthy humans and cancer patients. In immunocompetenthealthy subjects, a single booster injection with mature DCs couldenhance not only the frequency, but also the functional avidity of theCD8+ T-cell response. For these reasons, DCs (especially mature ones)are currently extremely promising adjuvants for induce potent T-cellresponses against tumors and infections in humans.

One precondition for the use of DCs in immunotherapy is the developmentof techniques which allow to produce a great number of DCs in culture,either from proliferating CD34+ precursor cells or fromnon-proliferating or little proliferating CD14+ monocytic precursorcells. DCs derived from monocytes are frequently used currently becausethey are easily prepared without any cytokine pretreatment of the donor,and because the resulting DC population is fairly homogeneous and bestcharacterized. For example, immature DCs can be prepared from adherentmonocytes in the absence of fetal calf serum (FCS) during a culture forusually six to seven days in (GM-CSF+IL-4), followed by maturation formostly one to three days, induced by autologous monocyte-conditionedmedium. To provide an effective cryoconservation for dendritic cells ortheir precursor cells has proven extremely difficult, except in thepresence of FCS (Taylor et al. (1990) Cryobiology 27, 269; Makino et al.(1997) Scand. J. Immunol. 45, 618). However, since FCS must not bepresent in vaccinations, it has still been necessary to prepare DCsfreshly for each DC vaccination, either from fresh blood, from freshleucapheresis products or from frozen PBMC (“peripheral bloodmononuclear cells”) aliquots from leucapheresis products (Thurner et al.(1999) J. Immunol. Methods 223, 1). Frozen PBMCs also have thedisadvantage that following the thawing the cells must first be culturedfor several days to differentiate them into DCs. Lewalle et al. (2000)J. Immunol. Methods 240, 69-78, disclose a method for the freezing ofimmature DCs, but only obtain poor yields of surviving cells (p. 71, topof left column). The freezing of immature dendritic cells prepared frommonocytes by means of GM-CSF and IL-4 has also been disclosed in WO99/46984.

SUMMARY OF THE INVENTION

Therefore, one object of the present invention is to provide acomposition, such as a vaccine, which contains mature DCs, that can bestored for extended periods of time without a substantial loss inactivity, and that can be administered within a short time when needed.

The DCs employed according to the invention are preferably derived fromthe same patient who is treated with the vaccine (autologous).Alternatively, the DCs may also be derived from other patients(allogenic), for example, from leucapheresates from normal volunteers,as described, for example, in Thurner et al. (1999) J. Immunol. Methods283, 1-15.

Surprisingly, it has been found that only fully matured DCs (hereinafteralso briefly referred to as “mature DCs”), which are obtainable byculturing in the presence of a suitable “maturing cocktail” withspecific maturing stimulants, will survive to a high percentage afterfreezing without FCS and thawing, and have immunostimulant activitieswhich are comparable to those of freshly prepared mature DCs. During theculturing with the maturing cocktail, the immature DCs aredifferentiated into mature DCs. A particularly preferred maturingcocktail contains interleukin-1β (IL-1β), interleukin-6 (IL-6),prostaglandin E₂ (PGE₂) and “tumor necrosis factor α” (TNFα).

Thus, the present invention relates to

(1) a method for the preparation of ready-for-use cryoconserved maturedendritic cells, comprising

a) providing immature dendritic cells (DC);

b) culturing the immature DCs in a culture medium containing a maturingcocktail with one or more maturing stimulants to obtain mature DCs;

c) freezing the mature DCs in a freezing medium which does not containany heterologous serum;

(2) a method as defined above under (1) which is suitable for thepreparation of a vaccine from mature cryoconserved DCs;

(3) frozen mature antigen-loaded dendritic cells, especially thosedendritic cells which are obtainable by the method (1);

(4) a vaccine comprising dendritic cells as defined in (3); and

(5) a method for finding advantageous conditions for the freezing ofDCs, comprising the steps of:

a) providing immature DCs;

b) performing different cultures with the immature DCs in the presenceof different maturing stimulants;

c) then culturing the DCs in medium with or without cytokines, culturingwithout cytokines being preferred;

d) determining the fraction of living cells after at least 1 day ofculture in said medium with or without cytokines; and

e) establishing that maturing stimulant which gave the highest survivalrate.

In methods (1) and (2) of the invention, immature DCs are firstcultured, a suitable maturing cocktail with one or more maturingstimulants being added to the culture medium. Suitable maturingstimulants include IL-1, such as IL-1β etc., IL-6, TNF-α,prostaglandins, such as PGE₂ etc., IFN-α, lipopolysaccharides and otherbacterial cell products, such as MPL (monophosphoryllipid A),lipoteichoic acid etc., phosphorylcholine, calcium ionophores, phorbolesters, such as PMA, heat-shock proteins, nucleotides, such as ATP etc.,lipopeptides, artificial ligands for Toll-like receptors,double-stranded RNA, such as poly-I:C etc., immunostimulant DNAsequences, CD40 ligand etc. According to the invention, it isparticularly preferred for said culture medium or maturing cocktail tocontain IL-1β, IL-6, PGE₂ and TNFα, or for said maturing cocktail to bemonocyte-conditioned medium (MCM) or MCM supplemented with PGE₂. In apreferred embodiment of methods (1) and (2), the DCs can be loaded withantigen during or after the culturing step.

After maturing and optionally loading with antigen, the DCs are frozenin freezing medium which does not contain any heterologous serum, suchas FCS. “Heterologous serum” within the meaning of this application is aserum which is not derived from the human species. However, according tothe invention, it is possible to use both autologous serum or plasma(which is derived from the same human as the DCs) and allogeneic serumor plasma (which is derived from a different human than for the DCs).

Immature DCs within the meaning of this application are CD83 (and/orp55/fascin and/or DC-LAMP) negative (or only weakly positive and/or to alow percentage) leukocytes which express only low amounts, as comparedto mature DCs, of class-I and Class-II MHC as well as adhesion orcostimulatory molecules (e.g., in particular, CVD86, CD80, CD40), whichwill differentiate into mature DCs upon a suitable maturing stimulant.

Mature DCs within the meaning of this application are leukocytes whichhave developed from immature DCs under the action of a maturingstimulant, exhibit an enhanced expression of CD83 (and/or p55/fascinand/or DC-LAMP) and of class-II and class-I MHC molecules and adhesionor costimulatory molecules, especially of CD86, CD80, CD40. Further, themature DCs exhibit a clearly increased T-cell stimulatory capacity ascompared to immature DCs (e.g., in contrast to immature DCs, theyexhibit a clear stimulatory activity in allogenic MLR even at a DC:Tratio of about ≦1:100); in addition, one characteristic of the matureDCs within the meaning of this application is the fact that these DCsare stable, i.e., will keep their properties of a mature phenotype and astrong T-cell stimulatory capacity even if cultured in the absence ofcytokines for 1-2 days or longer. In contrast, DCs which have not yetfully matured are not stable and will differentiate into immature DCsor, e.g., into adherent macrophages.

The immature DCs can be provided from various known sources. Theprecursor cells of the immature DCs are usually non-proliferatingCD14-positive mononuclear cells (PBMCs; monocytes) or proliferatingCD34-positive cells. For example, PBMCs can be isolated fromleucapheresis products. The PBMCs can be differentiated into immatureDCs as described (Thurner et al. (1999) J. Exp. Med. 190, 1669). Thus,the PBMCs are cultured in the presence of IL-4 (or IL-13; Romani et al.(1996) J. Immunol. Methods 196, 137-151) and GM-CSF(“granulocyte-macrophage colony stimulating factor”). From CD14-positivemonocytes, immature DCs can also be prepared by culturing in thepresence of GM-CSF and IFNα (Santini et al. (2000) J. Exp. Med. 191,1777-1788). From CD34-positive cells, immature DCs can be obtained byculturing in the presence of GM-CSF, SCF (“stem cell factor”) and TNFα.The immature dendritic cells may also be obtained directly from freshblood, such as described in Nestle et al. (1998) Nat. Med. 4, 328.Preformed CD11c+ and CD11c− DCs (O-Doherty et al. (1994) Immunology 82,487-493) and M-DC8-DCs (Schakel et al. (1999) Pathobiology 67, 287-290)also exist in blood. These DCs may also be isolated from the blood andfurther used in the method according to the invention.

The preferred concentrations of the various maturing stimulants in theculture medium are within a range of from 0.1 ng/ml to 100 μg/ml,preferably from 1 ng/ml to 10 μg/ml. For the particularly preferredmaturing cocktail of the present invention, the concentration of thematuring stimulants is from 0.1 to 100 ng/ml of IL-1β, from 0.1 to 100ng/ml of IL-6, from 0.1 to 10 μg/ml of PGE₂, and from 0.1 to 100 ng/mlof TNFα (most preferably, the concentration of these special maturingstimulants is approximately 10 ng/ml of IL-1β, 10 ng/ml of IL-6, 1 μg/mlof PGE₂, and 10 ng/ml of TNFα). The concentrations stated are the finalconcentrations of the substances in the cell culture medium in which theDCs are cultured. One possibility of maturing the DCs in the presence ofthe mentioned active substances is to culture the immature DCs in thepresence of monocyte-conditioned medium (MCM). MCM contains IL-1β, IL-6,PGE₂ and TNFα. It can be obtained by culturing PBMCs in medium withoutcytokines, as described, for example, in Romani et al. (1996) J.Immunol. Methods 196, 137. When the DCs are matured by MCM, the culturemedium contains from 1 to 100%, preferably from 5 to 25%, of MCM. Inanother embodiment of the invention, the maturing of the DCs is effectedby the addition of MCM and a defined amount of PGE₂. Namely, it has beenfound that variations in the capability of MCM to mature DCs in the bestpossible way can be counterbalanced by the addition of PGE₂ (preferablyin the concentrations as stated above). However, according to theinvention, it is preferred to mature the DCs by the addition of definedoptimum amounts of the active substances IL-1β, IL-6, PGE₂ and TNFα.This means that the maturing composition is prepared from purifiedformulations of the individual active substances. Better results areachieved thereby as compared with the maturing with MCM or MCM+PGE₂.IL-1β, IL-6, PGE₂ and TNFα are available is GMP (“good manufacturingpractice”) grade. Usually, the maturing is effected by cultivation inthe presence of the substances mentioned for at least one hour,preferably 2 hours and more preferably at least 6 hours. Particularlypreferred according to the present invention are maturing times of from6 to 96 hours, preferably from 18 to 36 hours (when leucapheresates areused as the starting material) or from 36 to 60 hours (when fresh bloodor buffy coat is used as the starting material).

According to the invention, the DCs are loaded with at least one antigenor an antigen-antibody complex during and/or after the maturing stepmentioned. According to the invention, the antigen loading can beeffected during or after the freezing. If it is effected after freezing,the DCs are loaded with antigen only after thawing. However, preferredis loading with antigen prior to the freezing of the DCs. This has theadvantage that the DCs are ready for use immediately after thawing.

Antigens within the meaning of this application are proteins, proteinfragments, peptides and molecules derived therefrom (e.g., glycosylatedcompounds or compounds having other chemical modifications), but alsothe nucleic acid encoding them, viruses, whole prokaryotic or eukaryoticcells or fragments thereof, or apoptotic cells. “Loading with antigen”means a process which causes MHC molecules on the cell surface of theDCs to “present” peptides, i.e., the peptides form a complex with theMHC molecules. While a direct loading of the MHC molecules. is possiblethrough specific peptides, proteins (or protein fragments) must first beprocessed, i.e., first taken up by the cell. After intracellularcleavage of the proteins and loading of MHCs with peptide, MHCII-peptidecomplexes are presented on the cell surface. Suitable antigens primarilyinclude proteins or protein fragments. The proteins may be of nativeorigin or have been prepared recombinantly. Due to the maturing andloading with protein antigen, MHCII-peptide complexes which comprise thepeptide fragments of the antigen added form on the cell surface. Theconcentration of the protein antigen in the culture medium is usuallyfrom 0.1 to 100 μg/ml, preferably from 1 to 50 μg/ml, most preferredfrom 1 to 10 μg/ml.

If a protein (i.e., a polypeptide having more than 50 amino acidresidues) is employed as the antigen, the loading should preferably beeffected during the maturing of the DCs. This causes a particularlyeffective presentation of the antigen fragments by MHCII molecules. Theprotein (fragment) need not be present during the whole maturing period,but at least for part of the maturing period, both the maturingcomposition and the protein (fragment) should be simultaneously present.Examples of protein antigens include KLH (keyhole limpet hemocyanin) asa widely used positive control antigen. MAGE-3 protein as an example ofa tumor antigen, and hepatitis B surface antigen (HBsAg) or HIV-1 gp-160protein as examples of a protein appropriate for the treatment of viraldiseases.

When the loading is performed with “short polypeptides” or proteinfragments (e.g., short polypeptides with up to 50 amino acid residueswhich exactly fit into the MHC molecules on the DC surface), loadingafter maturing, namely prior to freezing or after rethawing, is alsopossible in addition to the above mentioned loading during maturing. Insuch a loading after maturing, the short polypeptides are added towashed DCs.

Another possibility of loading with antigen is the addition of apoptoticor lysed cells to the culture medium. The added cells can then bephagocytosed by the DCs. This method has the advantage that, in additionto MHC class II molecules, class I molecules can also be effectivelyloaded. For example, it has been shown that presentation also occurs onMHC class I molecules even when proteins (especially of particulatenature) are added. For example, instead of culturing the DCs in thepresence of Mage-3 tumor protein or peptide, cell fragments (of necroticor apoptotic tumor cells) which contain Mage-3 can be added to the DCs.

DCs may also be loaded with antigen by fusing DCs, for example, withtumor cells. Such a fusion can be achieved with polyethylene glycol orelectroporation.

In another embodiment of the invention, the DCs are loaded with antigenby being transfected or virally infected. Nucleic acids coding forantigens are thereby introduced into the DCs or induced to expression.For example, a DNA or RNA transfection or infection with adenovirusescan be performed in a suitable way. Loading of the MHC class I moleculesis also possible thereby. For example, RNA prepared from tumor cells canalso be transfected. For transfection, usual methods, such aselectroporation, can be employed.

As set forth above, specific antigen peptide (i.e., “shortpolypeptides”) can be added during the maturing period or after thematuring period for directly loading MHC molecules of class I or II. Thepeptide antigen may be added to the cells before the maturingcomposition is added, but preferably, it is added simultaneously orthereafter. The antigen loading is preferably performed for at least 10minutes, more preferably for 1 to 24 hours, most preferably for 3 to 12hours.

The peptides (i.e., “short polypeptides”) usually have at least 8 aminoacids. Preferably, such peptides have from 8 to 12 amino acids (MHCI) orfrom 8 to 25 amino acids (MHCII). The concentration of the peptide inthe culture medium for loading is usually from 0.01 to 1000 μM,preferably from 0.1 to 100 μM, most preferably from 1 to 20 μM.

All peptides which can be presented by MHC molecules can be consideredas peptides to be employed. Preferred are peptides which are derivedfrom proteins derived from pathogens. These may also be peptides whichexhibit variations of the naturally occurring amino acid sequence. Suchvariations are usually one or two amino acid substitutions. Examples ofpeptide antigens are the influenza matrix peptide (IMP) having the aminoacid sequence GILGFVFTL (SEQ ID NO: 1) or the Melan-A-analogue peptidehaving the sequence ELAGIGILTV (SEQ ID NO: 2). Examples of otherpossible peptides are represented in FIG. 26.

In addition to “peptide/protein pulsing”, “peptide/protein transloading”may also be performed to load DCs with antigen.

As stated above, the DCs may also be loaded with antigen-antibodycomplexes. Suitable antigens for this purpose include all the antigensmentioned above. “Antigen-antibody complexes” according to the presentinvention are complexes of such antigens with suitable antibodies, e.g.,antigen-IgG or antigen-IgE complexes. The loading of DCs with suchantigens is described in Reynault, A. et al., J. Exp. Med. 189(2):371-80 (1999), which is included herein by reference.

After the maturing and optionally antigen loading of the DCs, the matureDCs can be frozen in freezing medium. In addition to the serumcomponent, the freezing medium may additionally contain one or morecryoprotectants (e.g., from 0.1 to 35% (v/v), preferably from 5 to 25%(v/v)). Suitable cryoprotectants preferably include the followingcompounds: DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol,albumin, choline chloride, amino acids, methanol, acetamide, glycerolmonoacetate, inorganic salts etc. The preferred cryoprotectant is DMSO,which is preferably contained in the freezing medium in a concentrationof from 5 to 25% (v/v), more preferably from 10 to 20% (v/v), mostpreferably about 10% DMSO. The freezing medium may contain anon-heterologous serum component, preferably in a concentration of from2 to 90% (w/v), preferably from 5 to 80% (w/v), wherein human serumalbumin, preferably in a concentration of from 10 to 30% (w/v), isparticularly preferred. More preferably, however, the freezing mediumcontains autologous serum or plasma instead of human serum albumin. Itmay also contain human allogenic serum or pool serum. Also, the freezingmedium may contain as an additive one or more polyol compounds derivedfrom carbohydrates, especially those selected from glucose, dextrane,sucrose, ethylene glycol, erythritol, D-ribitol, D-mannitol, D-sorbitol,inositol, D-lactose etc., preferably in a concentration of from 2 to30%, more preferably from 5 to 10%, most preferably about 5% (w/v). Themost preferred freezing medium is pure autologous serum with about 10%DMSO and about 5% glucose. Usually, the cells are centrifuged off priorto freezing to be concentrated and taken up in freezing medium.

The preferred concentration of cells in the freezing medium is from 1 to100×10⁶ cells/ml, the most preferred concentration being from about5×10⁶ cells/ml to 20×10⁶ cells/ml.

It has also been found that the survival rate of DCs is increased bycontacting the DCs with an anti-apoptotic molecule prior to freezing orafter thawing. Therefore, in a preferred embodiment of the methodaccording to the invention, The DCs are contacted prior to freezing orafter thawing with a molecule capable of inhibiting apoptosis. Preferredanti-apoptotic molecules include CD40 ligand (CD40L) (Morris et al.(1999) J. Biol. Chem. 274: 418), TRANCE (Wong et al. (1997) J. Exp. Med.186: 2075) or RANKL (Anderson et al. (1997) Nature 390: 175).Preferably, at least one of the molecules is added to the culture mediumprior to freezing or after thawing of the DCs for at least 10 min,preferably at least 1 hour, more preferably at least 4 hours. Preferredconcentrations in the culture medium are 1 ng/ml to 5 μg/ml(RANKL/TRANCE) and 1 ng/ml to 1 μg/ml (CD40L). Particularly preferredconcentrations are from 0.5 to 1 μg/ml (RANKL/TRANCE) and from 0.1 to0.5 μg/ml (CD40L).

In a preferred embodiment of the method, the DCs are further cultured inthe presence of immunosuppressive interleukin-10 (IL-10) or otherimmune/maturing modulators, such as vitamin D3 and its derivatives,fumaric acid and derivatives thereof, such as esters etc., mycophenolatemofetil etc. Cells thus treated are not employed for the stimulation ofthe immune system, but rather a tolerance against particular antigens isto be induced. The concentration of these modulators and of IL-10 in themedium is from 1 to 1000 ng/ml, preferably from 10 to 100 ng/ml.

According to embodiment (4), the present invention also relates to avaccine obtainable by the method according to the invention. In additionto the mature antigen-loaded DCs, the vaccine according to the inventionmay further contain pharmaceutically acceptable adjuvants. After thethawing of the vaccine according to the invention, various substancesmay be added which are pharmaceutically acceptable and advantageous foradministration.

According to embodiment (3), the invention also relates to frozen matureantigen-loaded dendritic cells. These cells according to the inventionmay be part of a pharmaceutical composition which contains usualadditives suitable for the administration to humans, in addition to thecells.

The present invention for the first time provides a vaccine whichcontains mature antigen-loaded DCs and can be frozen. An essentialadvantage of the invention is that the survival rate of the DCs afterthawing is very high. After the thawing of the frozen DCs, more than75%, preferably more than 85%, of surviving DCs are usually obtained,based on the number of frozen DCs. Such high survival rates of thawedDCs have not been achieved previously without the addition of FCS.However, this is an important precondition for achieving an effectiveimmunostimulation. A substantial advantage of the freezing of matureantigen-loaded DCs is that multiple aliquots of a vaccine can beprepared and frozen. Thus, the vaccine is immediately available whenneeded and can be applied without lengthy culturing steps within a veryshort time. By loading the mature DCs with the antigen only after thefreezing and rethawing, it is possible to load the DCs variablydepending on the respective circumstances. For example, when anoverstimulation or allergy against one of the peptides employed forvaccination is developing, this peptide can be omitted in the loading.

Surprisingly, it has been found that, for finding suitable methods forthe freezing of DCs, it is not sufficient to vary the immediate freezingparameters, such as freezing medium and cooling rate, and to determinethe survival rate immediately after thawing. This immediate survivalrate not necessarily corresponds to the survival of freshly prepared DCsduring several days of culture in the absence of cytokines.

Rather, the maturing process or the maturing stimulant employed prior tofreezing also play an important role to the survival of the cells. Thus,the invention is also based on the recognition that the maturingstimulant according to the invention not only leads to a full DCmaturation, but also yields DCs which can survive better than DCs whichhave been matured by other stimulants. To date, the maturing stimulantwas not considered at all in view of the freezing.

It has also been found that an excellent method for optimizing theprocess for the freezing of DCs is to mature DCs by means of differentmaturing stimulants and then to test the survival after culture incomplete medium without any addition of cytokine as a read-out. Thematuring stimulant which induces the most viable DCs is used for thepreparation of DCs which are then frozen.

Therefore, another aspect of the invention is a method for findingadvantageous conditions for the freezing of DCs wherein immature DCs areprovided and cultured in the presence of various maturing stimulants,the cells are subsequently cultured in medium with or without cytokines,preferably without cytokines, the fraction of surviving cells isdetermined after at least one day of culture in a medium with or withoutcytokines, and finally that maturing stimulant which gave the highestsurvival rate of DCs is determined. This maturing stimulant is then usedfor the preparation of DCs which are frozen. Preferably, the number ofliving cells is determined only after at least 2 days of culture in amedium without cytokines, most preferably after at least 3 days.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the influence of the cell concentration in the freezingvessels on the yield of DCs after thawing. Mature DCs were prepared fromleucapheresis products from healthy adults, wherein adherent monocyteswere first converted into immature DCs by six days of culture inGM-CSF+IL-4, followed by maturing for one day by a four-componentcocktail consisting of TNFα+IL-1β+IL-6+PGE₂ (see Example 1). Mature (day7) DCs were frozen at a freezing rate of −1° C./min in pure autologousserum+10% DMSO+5% glucose at different cell densities (number ofDCs/ml). After the thawing of the mature DCs, the yields of living DCs(stated as the percent fraction of the number of frozen DCs) weredetermined immediately (=d7) and after several days of culture (after 1day=d8, after 2 days=d9, etc.). The latter determination was performedin complete medium, but without the addition of GM-CSF or IL-4, sincethis hard “wash-out test” is an established method for determiningwhether DCs have actually been fully matured and are stable. Freezing at10×10⁶ mature DCs/ml yielded the best results (p<0.05 on day 7, p<0.01on all other days; n=3).

FIG. 2 shows the influence of various freezing media on the DC yieldafter rethawing. DCs were prepared as described in the legend of FIG. 1,and aliquots were frozen at a concentration of 10×10⁶ DCs/ml in HSA+10%DMSO, in autologous serum+10% DMSO, and in autologous serum+10% DMSO+5%glucose (final concentrations). The DC yields after freezing andrethawing were determined as described for FIG. 1. Freezing inautologous serum+10% DMSO+5% glucose yielded the best results (p<0.05 onday 8; n=4).

FIG. 3 shows the survival rate of fresh DCs in comparison with that offrozen and rethawed DCs in “wash-out tests”. Mature d7 DCs were preparedas described for FIG. 1, and then freshly prepared as well as frozen(−1° C./min in pure autologous serum+10% DMSO+5% glucose at a celldensity of 10×10⁶ DCs/ml) and rethawed after more than 3 hours ofstorage in the gas phase of liquid nitrogen) DCs were cultured forseveral days in medium without cytokines (“wash-out test”) as describedin FIG. 1. The yields of living DCs are stated as percent fractions ofthe total number of DCs sown into the wells on day 7. There was nostatistically significant difference between freshly prepared andfrozen/thawed DCs (n=4).

FIG. 4 shows that the freezing and rethawing does not change thecharacteristic morphology of mature DCs. Mature d7 DCs prepared asdescribed for FIG. 1 were either immediately further cultured foranother 2 days or were further cultured after freezing, at least 3 hoursof storage in the gas phase of liquid nitrogen and rethawing. Even after48 hours of further culture in the absence of cytokines (“wash-outtest”), the non-frozen (left) and frozen/rethawed (right) DCs remainedstable non-adherent cells.

FIG. 5 shows that the freezing and rethawing does not change thecharacteristic phenotype of mature DCs. Mature d7 DCs freshly preparedas described for FIG. 1 were phenotyped by a FACS analysis (fresh DC,day 7). Aliquots were frozen, stored for at least 3 hours in the gasphase of liquid nitrogen, rethawed, and subsequently their phenotype wasdetermined immediately (DCs frozen/rethawed d7) or after another 3 daysin culture ((DCs frozen/rethawed d10) in the absence of cytokines(“wash-out test”). Frozen/rethawed mature d7 DC maintained thecharacteristic phenotype of mature DCs (CD14−, CD83 homogeneously ++)even after the removal of cytokines and further culture for 3 days.

FIG. 6 shows that the freezing and rethawing does not change thestimulatory capacity of mature DCs in the primary allogenic MLR (“mixedleucocyte reaction”). Mature d7 DCs were prepared as described for FIG.1, and the allostimulatory potency of freshly prepared non-frozen DCswas compared with that of an aliquot of these DCs which had been frozenand rethawed (after 3 hours of storage in liquid nitrogen).

FIG. 7 shows that the freezing and rethawing does not change the abilityof mature DCs to induce a strong IMP-specific CTL response. Freshlyprepared or frozen/rethawed HLA-A2.1+ mature d7 DCs were loaded withinfluenza matrix peptide (=IMP) GILGFVFTL (SEQ ID NO: 1) or leftuntreated and cultured with autologous CD8+ T cells (T:DC ratio=10:1)without adding any cytokines. Additionally, purified CD8+ T cells werecultured without DCs±addition of IMP. After 7 days, the T cells wereharvested and examined for their cytologic activity using a standard⁵¹Cr release assay. T2 cells pulsed without or with 10 μg/ml of IMPserved as the target cells.

FIG. 8 shows that the freezing and rethawing does not change the abilityof mature DCs to induce a strong IMP-specific CD8+ T cell response(HLA-A2.1/peptide tetramer analysis). Freshly prepared orfrozen/rethawed HLA-A2.1+ mature d7 DCs were loaded with IMP (in thecase of frozen/rethawed cells, prior to freezing or after thawing) orleft untreated and co-cultured for 7 days with autologous non-adherentfractions of PBMCs (PBMC:DC ratio=30:1) without adding any cytokines,harvested and double-stained with HLA-A2.1/IMP tetramers (X axis) andanti-CD8 (Y axis). The expansion of IMP-peptide-specific CD8+ T cells(the percentage of tetramer-binding CD8+ T cells is stated in theFigure) is comparable for fresh and frozen DCs. Loading DCs prior tofreezing or after thawing does not make a difference.

FIG. 9 shows that a contact with CD40L further improves the survivalrate of thawed DCs. Mature d7-DCs were prepared as described for FIG. 1.After thawing, soluble primary CD40L (100 ng/ml) was added for 4 hours,then the DCs were washed and cultured for several days in medium withoutcytokines (“wash-out test”) as described in the legend for FIG. 3. Theyields of living DCs (stated as the percentage of frozen DCs) weredetermined immediately (=d7) and after several days of culture (after 1day=d8, after 2 days=d9, etc.). CD40L treatment of DCs yielded animproved survival rate, especially beyond day 2 of the further culture(p<0.01 on day 10 and day 11; n=5).

FIG. 10 shows that DCs can be successfully loaded with protein antigenprior to freezing. DCs were pulsed in their immature stage by adding themodel antigen tetanus toxoid (TT) (10 μg/ml) on day 6. Mature DCs werethen harvested on day 7 and frozen as described in the legend for FIG.3. Frozen TT-pulsed DCs were stimulatory like freshly prepared TT-pulsedmature DCs.

FIG. 11 shows that DCs can be successfully loaded with protein antigenprior to freezing. Frozen/thawed (see FIG. 3) HLA-A2.1+ mature d7 DCswere loaded with Melan-A-analogue peptide ELAGIGILTV (SEQ ID NO: 2)either prior to freezing or after thawing or left untreated andco-cultured for 7 days with autologous non-adherent fractions of PBMCs(PBMC:DC ratio-20:1) without adding any cytokines, harvested anddouble-stained with HLA-A2.1/Melan-A tetramers (X axis) and anti-CD8 (Yaxis). The expansion of Melan-A-peptide-specific CD8+ T cells (thepercentage of tetramer-binding CD8+ T cells is stated in the Figure) iscomparable for frozen DCs which had been loaded prior to freezing orafter thawing. See also FIG. 8.

FIG. 12 shows the induction of a helper cell type 1 response against KLHin patients who were vaccinated as described in Example 6.

FIGS. 13-15 show the results of Example 7.

FIG. 16 shows a schematic representation of the experimental set-up ofExample 8. The results of Example 8 are summarized in FIGS. 17 and 18.

FIG. 17A shows the total number of DCs after loading with tumor celllysate or necrotic tumor cells and subsequent cryoconservation.

FIG. 17B shows the allostimulatory potency of DCs after loading withtumor cell lysate or necrotic tumor cells and subsequentcryoconservation.

FIG. 17C shows the result of the FACS analysis after tumor loading andcryoconservation.

FIG. 18A shows the result of the determination of the total number ofDCs after loading with apoptotic tumor cells and subsequentcryoconservation.

FIG. 18B shows the allostimulatory potency of DCs after loading withapoptotic MEL 526 melanoma cells and subsequent cryoconservation.

FIG. 18C shows the results of the FACS analysis of MAGE-1/HLA-A1 Agexpression on Mage-1 peptide-pulsed DCs prior to and aftercryoconservation.

FIG. 18D shows a comparison of the expression of MAGE-1/HLA-A1 complexes(determined by means of antibodies against MAGE-1/HLA-A1 complex) priorto and after cryoconservation on DCs which had been loaded in differentways.

FIG. 19 shows a schematic representation of the experimental set-up ofExample 9 in which adenovirus-infected dendritic cells with or withoutcryoconservation are compared. The results of Example 9 are summarizedin FIGS. 20 to 23.

FIG. 20 shows the recovery of adenovirus-infected dendritic cells aftercryoconservation. The recovery rate of adeno-GFP infected DCs afterfreezing and rethawing is comparable with that of mock-treated(non-transfected) DCs.

FIG. 21: CD4 T-cell stimulating activity of adenovirus-infecteddendritic cells after cryoconservation. It can be shown that thecryoconservation does not change the allostimulatory activity ofadenovirus-infected DCs.

FIGS. 22A and 22B show that a similar phenotype is observed inadenovirus-infected DCs with or without cryoconservation.

FIGS. 23A and 23B show a phenotype of adenovirus-infected DCs with orwithout cryoconservation.

FIG. 24 shows the schematic course of the experiment of Example 10 ascompared with RNA-electroporated DCs with or without cryoconservation.The results are summarized in FIGS. 25 to 27.

FIG. 25 shows that the recovery after cryoconservation ofEGFP-RNA-electroporated DCs is similar to that in the controlexperiment.

FIG. 26 shows that the cryoconservation does not change theallostimulatory capacity of EGFP-RNA-transfected DCs.

FIG. 27 shows that the phenotype of RNA-transfected DCs is similar tothat in the control experiment.

FIG. 28 shows MHC class I and II restricted melanoma and influenza viralpeptides which can be employed in the method of the present invention.Abbreviations: AS=amino acids, NT=nucleotides, NP=nucleoprotein,ana*=analogue peptide

^(a) Most frequent DR4 subtype, approximately 80%

^(b) Most frequent DR4 subtype, approximately 80%

^(c) To date, 30 different HLA DR13 alleles have been described.Restriction for DRB1*1301 and DRB1*1302, which together make up 80% ofthe DR13 alleles, was shown. It is possible that other DR13 alleles alsopresent the peptide.

^(d) The epitope is presented less effectively by this second HLA DP4allele as compared to the allele DPB1*0401.

DETAILED DESCRIPTION OF THE INVENTION

The following Examples are intended to further illustrate the invention,but without any limitation thereto.

Example 1

The survival rates of freshly prepared immature DCs and of DCs maturedby different maturing stimulants were determined.

The following different maturing stimulants were tested:

Monocyte-conditioned medium, prepared and used as described (Thurner etal. (1999) J. Immunol. Methods 223, 1);

10 ng/ml TNFα;

10 ng/ml TNFα+1 μg/ml PGE₂;

10 ng/ml TNFα+10 ng/ml IL-1β+100 U/ml IL-6+1 μg/ml PGE₂ (“maturingcocktail”);

up to 1.0 μg/ml of LPS of Salmonella abortus equi;

double-stranded RNA (poly-I:C, 20 μg/ml);

from 50 to 1000 ng/ml CD40L.

Preparation of monocyte-derived DCs from PBMCs: As a complete medium,RPMI 1640 with 20 μg/ml gentamicin, 2 mM glutamine and 1%heat-inactivated (56° C., 30 min) autologous human plasma was used.Leucapheresis products were prepared as monocyte separation productsfrom healthy cytapheresis donors as described (Thurner et al. (1999) J.Immunol. Methods 223, 1). Peripheral blood mononuclear cells (PBMCs)were then isolated by centrifugation in lymphoprep, and DCs wereprepared from plastic-adherent fractions of the PBMCs as described(Thurner et al. (1999) J. Immunol. Methods 223, 1). For this purpose,immature DCs were obtained by a culture in complete medium withrecombinant human GM-CSF (800 U/ml) and IL-4 (500 U/ml). On day 6, thedifferent maturing stimulants mentioned above were added to the DCs, andon day 7, the mature DCs were harvested and further cultured for anothertwo days in the absence of cytokines. The survival rates after two daysof culture in percent of the sown DCs were:

38.25% for immature DCs; 76.2% for (IL-1β+IL-6+PGE₂+TNFα)-matured DCs;

13.5% for TNFα-matured DCs;

37.0% for (TNFα+PGE₂)-matured DCs;

31.8% for (poly-I:C)-matured DCs; and

49.6% for CD40L-matured DCs (at 500 ng/ml).

The differences were statistically significant.

Example 2

Mature (day 7) DCs were frozen as follows: DCs were resuspended incryovessels at different concentrations (5, 20, 40, 60 and 100×10⁶ perml) either in pure autologous serum or in 20% human serum albumin (HSA;consisting of 1000 ml of electrolyte solution supplemented with 200 g ofhuman plasma proteins with at least 95% of albumin; DRK BlutspendedienstBaden-Wurttemberg, Baden-Baden, Germany). The DC suspension formed wasmixed 1:1 with the different freezing solutions described hereinafterand then immediately transferred into a 1.0 or 1.8 ml cryovessel.Immediately thereafter, the vessels were cooled down to −80° C. in a“Cryo Freezing Container” (Nalgene Cryo 1° C. Freezing Container,cooling rate −1° C./min) and finally transferred into the gas phase ofliquid nitrogen, where they were kept for up to seven months.

a) Freezing Media

HSA+DMSO±glucose (consisting of 20% HSA solution (see above)+10, 15, and25% (v/v) DMSO±glucose (Glukosteril 40%™, Fresenius, Germany, activeingredient: glucose monohydrate) at 2, 4, 6, 10, 20 or 30% (v/v));

serum+DMSO±glucose (pure autologous serum+20% (v/v) DMSO±glucose addedat 2, 4, 6, 10, 20 or 30%);

Erythrocyte Freezing Solution™ (Erythrocyte Freezing Solution™(Fresenius, Dreieich, Germany), consisting of 38% glycerol, 2.9%sorbitol and 0.63% sodium chloride in sterile water);

Cell Processing Solution™+DMSO (Fresenius, Dreieich, Germany, consistingof 6% hydroxyethylstarch in 0.9% sodium chloride, usually used for thesedimentation of erythrocytes)+10 or 15% (v/v) DMSO.

b) Thawing Conditions

For the thawing of frozen mature (day 7) DCs, the following four methodswere examined:

1. DCs were thawed in a water bath at 56° C., then incubated withoutwashing in 10 to 20 ml of ice-cold complete medium (supplemented with800 U/ml of GM-CSF and 500 U/ml of IL-4) in Teflon dishes (Rotilaboboxes, Roth, Karlsruhe, Germany) at 37° C. and 5% CO₂ for two hours,then harvested and centrifuged for 10 minutes at 150×g and 22° C.Subsequently, the cells were counted and again sown on Teflon dishes incomplete medium with GM-CSF and IL-4 (cell density 1×10⁶ per ml) andcultured over night. On the next day, the cells were harvested forfurther use.

2. Frozen mature (day 7) DCs were thawed as described under 1, and aftera resting period of two hours at 37° C. and 5% CO₂ on Teflon dishes, thecells were harvested (centrifugation for 10 min at 150×g and 22° C.) forfurther use.

3. Frozen mature (day 7) DCs were thawed as described under 1, and aftera resting period of two hours at 37° C. and 5% CO₂ on tissue culturedishes (Falcon Becton Dickinson Labware, New Jersey, USA), the cellswere harvested (centrifugation for 10 min at 150×g and 22° C.) forfurther use.

4. Frozen mature (day 7) DCs were thawed in a water bath at 56° C., thenadded to 10 ml of ice-cold “Hank's Balanced Salt Solution” (BioWhitaker) and immediately centrifuged for 12 min at 133×g and 4° C.Subsequently, the cells were harvested for further use.

c) Establishing of DC Survival Rate

The survival rate of frozen and rethawed DCs was examined by culturingin complete medium without the addition of GM-CSF and IL-4 over at least4 days (=“wash-out test”) and compared with the survival rate of DCswhich had been freshly prepared from non-frozen or frozen aliquots ofPBMCs as described (Thurner et al. (1999) J. Immunol. Methods 223, 1).The respective amounts of living DCs were determined by a cell counter(Cassy Cell Counter and Analyser System, Model TT, Schrfe System,Reutlingen, Germany; this system uses “pulse area analysis” and allowsthe determination of cell counts, cell size and volume as well aswhether living cells are present) and as a control also through standardtrypan blue staining.

First, the influence of the DC concentration was examined, whereinfreezing at 10×10⁶ mature DCs/ml yielded the best results. The result isshown in FIG. 1.

Further, the influence of the DMSO concentration (5 to 12.5% v/v finalconcentration) was examined. A change of the DMSO concentration had nosignificant effect on the survival rate of thawed DCs.

Further, it was examined whether HSA or pure autologous serum yieldedbetter survival rates, and whether an addition of glucose (finalconcentrations v/v of 1, 2, 3, 5, and 15%) improves the survival rate.The result is shown in FIG. 2. The survival rate of DCs after severaldays was reproducibly increased if HSA was replaced by autologous serum.An addition of glucose in a final concentration of 5% (v/v) furtherimproved the results.

Various commercially available freezing media, such as ErythrocyteFreezing Solution™ or Cell Processing Solution (Fresenius) were alsoexamined. However, these yielded worse results as compared with thefreezing media already tested.

Also, the various thawing conditions mentioned under b) were examinedfor minimizing the stress to which the cells are subjected afterthawing. None of the four methods tested showed a clear superiority overthe others.

Mature (day 7) DCs were prepared as described in Example 1 and frozenunder the following conditions:

Cooling rate 1° C. per min in pure autologous serum+10% DMSO+5% glucoseat a cell density of 10×10⁶/ml. After at least three hours of storage inthe gas phase of liquid nitrogen, the cells were thawed. After thawing,the percentage of living DCs was directly determined (=D7). Aliquots ofthe DCs were sown, and the survival rates were determined after up to 4days in culture in medium without cytokines (“wash-out test”) asdescribed in Example 1c) (n=20). The result is shown in the followingTable.

Yield of Frozen/Thawed DCs after Thawing (Day 7) and in “Washout Tests”(Days 8 to 11).

Days Yield in % 95% confidence interval 7 93.3 100.0-85.8  8 74.181.7-66.6 9 63.3 70.8-55.7 10 55.8 63.4-48.2 11 47.1 57.9-36.2

Example 3

Optimally matured and frozen DCs are equivalent to freshly prepared DCswith respect to survival rate and T-cell stimulatory activity.

a) First, it was examined whether the survival rate of frozen andrethawed DCs is comparable with the survival rate of freshly preparedDCs from the same donor. Thus, the “wash-out test” was used as describedin Example 2c). The result is shown in FIG. 3. It was found that thereis no difference between thawed DCs and freshly prepared DCs from thesame donor.

Also, the morphology and the phenotype of thawed DCs were compared withthose of freshly prepared DCs. The morphology of the cells was examinedwith an inverted-phase microscope (Leika DM IRB, Leika Mikroskopie andSysteme GmbH, Wetzlar, Germany) and recorded by photography. Thephenotype of the cell populations was examined by a series of monoclonalantibodies and examined on a FACScan device (Becton Dickinson, NewJersey, USA) as described (Thurner et al. (1999) J. Immunol. Methods223, 1). Dead cells were sorted out due to their light-scatteringproperties. The result is shown in FIGS. 4 and 5. Frozen and rethawedDCs keep their characteristic morphological properties and theirphenotype over several days.

b) Then, it was examined whether rethawed DCs also maintain theirfunctional properties.

1. Thus, it was examined whether rethawed DCs can induce primaryallogenic MLRs as effectively as freshly prepared, non-frozen matureDCs. This test was performed as described (Thurner et al. (1999) J.Immunol. Methods 223, 1). DCs were added in graded doses to 2×10⁵allogenic T cells per well in flat-bottomed 96-well plates andco-cultured for 4 to 5 days in RPMI 1640 (supplemented with gentamicin,glutamine and 5% allogenic heat-inactivated human serum (pool serum)),and the proliferation was determined by the addition of ³H-thymidine (4μCi final concentration/ml) for the last 12 to 16 hours of theco-incubation. The result is shown in FIG. 6. Frozen and rethawed DCsinduce primary allogenic MLRs as effectively as freshly prepared matureDCs.

2. It was also examined how effectively frozen and rethawed DCs caninduce cytotoxic T lymphocytes (CTLs). For this purpose, the inductionof IMP (influenza matrix peptide) specific CTLs by IMP-pulsed mature DCswas measured. This approach is specific for mature DCs when performed inthe absence of T cell assistance and exogenic IL-2. The induction ofCD8+ T cells specific for influenza matrix A2.1 peptide (IMP) or Melan-AA2.1 peptide was effected by the stimulation of purified CD8+ T cells(isolated from PBMCs by magnetic cell sorting/MACS with CD8 microbeadsaccording to the supplier's specifications, Miltenyi Biotec, BergischGladbach, Germany), or in other experiments of non-adherent PBMCfractions with DCs (prepared from autologous PBMCs from HLA-A2.1+donors)which were either unpulsed or pulsed with HLA-A2.1 restricted IMP(GILGFVFTL [SEQ ID NO: 1], 10 μM for 1 hour at 37° C. at 1×10⁶ DCs/ml ofcomplete medium) or Melan-A-analogue peptide (ELAGIGILTV [SEQ ID NO: 2],10 μM) at a DC/T ratio of 1:10 or 1:30 for 7 days without the additionof cytokines. CTLs were quantified by a standard lysis assay (Bhardwajet al. (1994) J. Clin. Invest. 94, 797) or by tetramer staining at 37°C. (Whelan et al. (1999) J. Immunol. 163, 4342). The target cells forthe standard 4-hour ⁵¹Cr-release assay, which was performed at differenteffector/target cell ratios, were IMP-pulsed (10 μg/ml for 1 hour at 37°C.) T2A1 cells, unpulsed T2A1 cells and K562 target cells (all.sup.51Cr-labeled). All experiments were performed with an 80 foldexcess of K562 cells in order to block the natural killer cell activity.The specific lysis in % was calculated by the formula (specificrelease−spontaneous release)/(maximum release−spontaneous release)×100.Soluble IMP and Melan A/HLA A2.1 tetramers were prepared, and theformation of T cells was analyzed by flow cytometry at 37° C. asdescribed (Whelan et al., 1999). 1 μl of tetramer (0.5 to 1 mg/ml) wasadded to 2×10⁶ cells in about 60 μl (volume remaining in the vesselafter centrifugation and pouring of the supernatant) of mediumconsisting of RPMI 1640 supplemented with gentamicin, glutamine and 5%allogenic heat-inactivated human serum (pool serum) for 15 min at 37° C.Subsequently, the cells were cooled down without washing and incubatedon ice for 15 min with a triply stained monoclonal antibody againsthuman CD8 (Caltag Laboratories, Burlingame, Calif.). After three washingsteps, the cells were analyzed on a FACScan device (Becton Dickinson).

The result is shown in FIGS. 7 and 8.

The freezing and thawing of DCs does not change the capability of matureDCs of inducing a strong IMP-specific CLT response. Also, the freezingand thawing does not change the capability of mature DCs of inducingstrong IMP-specific CD8+ T cell responses, as shown by theHLA-A2.1/peptide tetramer analysis.

These experiments show that, after the freezing and thawing, living DCswere obtained which are absolutely equivalent with freshly prepared DCs.

Example 4

To achieve an increased survival rate of the frozen and rethawed DCs,the following different anti-apoptotic stimulants were added to the DCsin different concentrations and at different times:

1. Recombinant murine or human trimeric TRANCE (Wong et al. (1997) J.Exp. Med. 186, 2075) at 100, 200, 500 ng/ml;

2. RANKL (Anderson et al. (1997) Nature 390, 175) at 10 ng/ml, 50 ng/ml,100 ng/ml and 1 μg/ml;

3. Trimeric soluble CD40L (Morris et al. (1999)J. Biol. Chem. 274, 418)at 50, 100 and 500 ng/ml.

The DCs were subjected to the different anti-apoptotic stimulants at 37°C. over night for the last 12-16 h of culture prior to freezing, for 4 hprior to freezing (cell density 1×10⁶ in complete medium with GM-CSF andIL-4), and also for 4 h after thawing (cell density 1×10⁶ in completemedium with GM-CSF and IL-4).

A brief exposure of thawed DCs to the anti-apoptotic stimulants was aseffective as the DC treatment prior to freezing and gave an increasedsurvival rate, which often became visible only after 3 days in the“wash-out test”, however. CD40L (FIG. 9) and TRANCE/RANKL (results notshown) yielded similar results. The results show that the addition ofCD40L or TRANCE/RANKL improves the survival rate of DCs beyond day 3.

Example 5

In order to examine whether DCs can be successfully loaded with antigenprior to freezing, DCs were loaded with tetanus toxoid (TT) (as anexample of a protein antigen) or with IMP (as a model peptide). Forpulsing with TT, DCs were prepared from fresh or frozen aliquots ofPBMCs from leucapheresis products (see above). TT was added to theimmature cells on day 5 at 10 μg/ml. Mature DCs were harvested on day 7and, either non-frozen or after freezing and thawing (after 4 h),examined for their capability of inducing TT-specific proliferativeresponses in PBMCs. Thus, graded doses of unpulsed and TT-pulsed DCswere added to PBMCs (10×10⁴/well) and pulsed with ³H-thymidine on day 5as described (Thurner et al. (1999) J. Immunol. Methods 223, 1). Forloading with IMP, DCs were pulsed with 10 μM peptide (for 1 h, 37° C.,1×10⁶ DCs/ml of complete medium) either prior to freezing or afterthawing. The capability of successfully presenting IMP was tested asdescribed above.

Frozen TT-pulsed DCs had the same stimulatory properties as freshlyprepared TT-pulsed DCs (FIG. 10). Both DCs loaded with IMP or Melan Aprior to freezing and those loaded after thawing stimulated IMP- orMelan-A-specific CTLs equally well (FIGS. 8 and 11). These results showthat it is possible to prepare frozen aliquots of mature DCs which havealready been loaded with antigen and can be used immediately afterthawing.

Example 6

With stage IV melanoma patients, a vaccination was performed withpeptide- and protein-loaded DCs prepared and antigen-loaded according tothe method shown herein. FIG. 12 shows the induction of a helper celltype 1 response against KLH in all vaccinated patients. Each of thepatients obtained a single subcutaneous delivery of 4 million mature DCswhich had been prepared from leucapheresates using GM-CSF andinterleukin-4 and the maturing composition as described in theapplication (e.g., FIGS. 1 and 3). In doing so, the control antigen KLHwas added in a concentration of 10 μg/ml simultaneously with thematuring composition, and the DCs were then frozen in portions. Prior tovaccination and 14 days after said single administration of 4 millionDCs, blood was removed and examined by a standard Elispot assay(addition of 10 μg/ml KLH to 500,000 PBMCs and measurement of the numberof cells producing interferon-.gamma. or IL-4; the background withoutthe addition of KLH is almost 0). It is found that neither interferon-γnor interleukin-4 is produced upon KLH presentation prior tovaccination, whereas 14 days after the single vaccination with theKLH-loaded DCs, T cells which produced interferon-γ, but did not produceinterleukin-4 or only minimally so were induced in all patients (incontrol experiments, it was shown by removing the CD4 cells from thePBMCs that the reactive cells are CD4-positive helper T cells).

Example 7

A patient from the study of Example 6 received several vaccinations withDCs, in which 4 million each of rethawed DCs were loaded with thepeptides stated in FIG. 13, 14 or 15 (i.e., only 1 peptide respectivelyper 4 million DCs) and injected subcutaneously. Respectively prior tothe first vaccination and 14 days after the respectively precedingvaccination and immediately before the next vaccination, blood wasremoved, and a standard Elispot assay was performed as described inThurner et al. (1999), J. Exp. Med. 190, 1669-1678, under Materials andMethods. The DCs used for the vaccination were prepared as stated in thelegend for FIGS. 8 and 11 and in Example 5, and loaded with therespective peptides only after thawing. As can be seen from FIGS. 13 to15, immunity against several class-I (FIGS. 13 and 14) and class-IIpeptides (FIG. 15) was induced. The characteristics of the peptidesemployed can be seen from Table I. It is to be noted that the HLA typeof the particular patient is HLA-A 2.1+ and HLA-A3+ as well as HLA-DR13+, HLA-DP4+ (but DR4−). FIG. 13 shows the induction of immunityagainst the influenza peptides NP-A3 and IMP-A2 upon a singlevaccination (No. 2 means the removal of blood and Elispot measurementbriefly before the administration of vaccination No. 2) and shows anincrease after another vaccination, but no change in EBV-A2 and IV-9-A2peptides, which were not used for vaccination. FIG. 14 shows aninduction of immunity against four class-I restricted tumor peptides,clearly and unambiguously against the peptide Melan A-A2.1. FIG. 15shows an induction of immunity against two class-II restricted tumorpeptides (Mage 3-DR13 and -DP4) but not against tyrosinase-DR4 and GP100 DR4 (this is a negative control since the patient was DR4-negative,and the DCs thus could not be loaded with these peptides).

Example 8 Cryoconservation of Dendritic Cells After Antigen Loading withTumor Cell Preparations Tumor Cell Lysates, Necrotic or Apoptotic TumorCells

Dendritic cells (DCs) can be generated in large amounts fromleucaphereses. A method for the cryoconservation of mature DCs has beendeveloped which allows the portioned use of these cells for vaccination.To date, the mature DCs were loaded prior to use with peptides whichcorrespond to the immunodominant sequences of tumor-associated antigens(TAA). In the experiments described here (see FIG. 16), it was examinedwhether immature DCs which were loaded with different tumor cellpreparations and subsequently matured can also be cryoconserved.

Tumor cell preparation: For preparing the tumor cell preparations, theMel526 melanoma cell line was used. Mel526 cells were washed in RPMI andtreated by repeated heatings at 57° C. and subsequent coolings in liquidnitrogen. Then, the cell material was disrupted by means of anultrasonic device. Since the heating/freezing cycles induce necrosis,this kind of tumor cell preparation which contains all cell componentsis referred to as necrotic cell material in the following. For obtaininglysate, we performed an ultracentrifugation after these steps to removecell components and effected purification of the proteins in a Centriconcentrifugation tube. According to the results of the Bradford analysis,we used the protein fraction having the higher activity, namely that of≧10 kDa. Apoptotic tumor cells were induced with a broad-range UVBirradiation device and verified with an Annexin V Test.

Generation of DCs: DCs were generated from leucaphereses according tothe technique used in experimental immunotherapy. PBMCs were plated inNunc Cell Factories and cultured with RPMI (1% autologousheat-inactivated plasma) supplemented with 1000 IU/ml GM-CSF and 500IU/ml IL-4. On day 5, the DCs, which were immature then, were used andloaded for 4 hours with the tumor cell preparations described in aconcentration of 1:1.

Loading: The loading was effected in a 5 ml polypropylene reactionvessel at 37° C. and 5% CO₂. After the loading, the DCs were plated in12 ml tissue culture dishes and cultured with a maturing cocktailconsisting of TNF-α, IL-1β, IL-6 and PGE₂ for 24 h.

Freezing/thawing: Half of the respectively loaded DCs were respectivelycryoconserved in 1.8 ml Nunc freeze vials at −80° C. for 3 hoursaccording to the method described (Feuerstein et al., J. Immunol.Methods 245: 15-29 (2000)). Thereafter, the cells were again thawedaccording to the method described and cultured for one hour in RPMImedium at 37° C. and 5% CO₂. Subsequently, this fraction of the DCs aswell as the non-frozen fraction were analyzed and used for furtherexperiments.

The experimental set-up described is shown as a flow chart in FIG. 16.

Cell count and viability: The total cell count and viability of the DCswere determined with trypan blue with a microscope after performance ofthe loading described and cryoconservation. Initially, 5×10⁵ DCs wereused. We found comparable cell counts and no difference in the viabilityof the loaded and cryoconserved DCs as compared with thenon-cryoconserved cells (FIGS. 17A, 18A). A reduction of the cell countby the loading, but not by the cryoconservation, especially withnecrotic cells, could be observed.

Functionality: For testing the functional capacity, a mixed-leucocytereaction test (MLR) was performed. The loaded DCs were employed in anallogenic MLR (4 days of incubation with allogenic leucocytes) and thenpulsed with radioactive thymidine (³H-thymidine) for 13 hours.Comparable allostimulatory potencies were obtained for cryoconserved andnon-cryoconserved loaded DCs (FIGS. 17B, 18B).

Phenotype: For evaluating the expression of the surface moleculesrelevant to antigen presentation and the functional condition of theDCs, a FACS analysis was performed with the corresponding antibodies. Acomparable surface expression pattern was found both for the differentloading methods and after cryoconservation (FIG. 17C).

Direct antigen detection: For the direct detection of tumoral antigen,an antibody was employed which recognizes MAGE-1 in an HLA-A1 context,i.e., the complex of Mage-1 peptide and the HLA-A1 molecule. Thedifferently loaded DCs were dyed with this antibody and analyzed in theFACS. When peptide-loaded (20 μg MAGE-1 peptide for 3 hours/ml) DCs wereused, it was found that after cryoconservation, a percentage ofMAGE-1/A1 antigen could be detected which was comparable to thatdetected without cryoconservation (FIG. 18C). From this experiment, itcan be concluded that the cryoconservation does not lead to a loss inantigen.

In a further experiment, the MAGE-1/A1 antibody was employed fordetermining the effectiveness of the different loading methods. With thetumor cell preparations (the melanoma cell line employed expresses theMage-1-antigen), a significant loading could be achieved which reachedabout 20% of the antigen density of the peptide pulsing (FIG. 18D). Theexpression of the MAGE-1/HLA-A1 complex before and after thecryoconservation could be detected in comparable quantities (calculatedas positivity against the background of unloaded DCs).

Conclusion: It could also be shown that the method of the presentinvention allows not only an effective cryoconservation of unloaded DCsor DCs loaded with peptide or protein (as shown in Examples 1-7), butalso a similarly effective cryoconservation of DCs loaded with (tumor)cell preparations (simple necrotic tumor cells, lysates prepared fromtumor cells, or apoptotic tumor cells). “Effectively” means that uponfreezing, i) the cell loss after thawing is ≦25% as compared withnon-frozen DCs, ii) the thawed DCs have a T cell stimulatory capacitycomparable to that of the non-frozen DCs (tested in allogenic MLR), andiii) the surface expression of antigens and ligands for T cell receptors(i.e., specific MHC peptide complexes) is retained after the freezingand thawing process (shown in a model by the direct detection of aparticular peptide/MHC complex, namely the MAGE-1/HLA-A1 complex, bymeans of a monoclonal antibody which specifically recognizes thiscomplex).

Example 9 Cryoconservation of Dendritic Cells after Antigen Loading byMeans of Adenoviral Transfection

Dendritic cells transfected with adenoviruses can be frozen by themethod according to the invention in such a way that the properties ofthe dendritic cells are comparable with those of non-transfecteddendritic cells. For this purpose, mature DCs were infected with anadenoviral vector (AD5) which contained a cDNA coding for the greenfluorescent protein (GFP) at a multiplicity of infection (MOI) of 500for 2 hours. After two washes, the cells were frozen at a concentrationof 10×10⁶ DCs/ml in HSA and 10% DMSO in 5% glucose (final concentration)and stored for 4 hours. After thawing, the viability of the cells wasdetermined by trypan blue exclusion. The recovery rate of viable cellsis stated in FIG. 20 as a percentage of frozen DCs.

Further, it could be shown that the allostimulatory activity ofadenovirus-infected DCs is not changed by cryoconservation. Thus, matureDCs were infected with adeno-GFP at an MOI of 500 and cryoconserved asdescribed above. After rethawing and a culturing period of 24 or 72hours, the dendritic cells were co-cultured with allogenic CD4+ T cells(2×10⁵ per well) under the conditions stated in FIG. 21. After 4 days,the cells were pulsed with [³H]-thymidine for 16 hours, and theincorporated radioactivity was determined. In FIG. 21, the averagevalues of three counts are stated with the corresponding standarddeviations. The values for T cells alone or DCs alone were always lessthan 1000 per min.

Further, mature DCs are cryoconserved with adeno-GFP at an MOI of 500 asdescribed above. After rethawing and the stated culturing time, thecells were counterstained using antibodies specific for CD83, CD25,CD86, CD80 followed by PE-conjugated goat/mouse IG (Fab′)₂ fragments.The results are shown in FIG. 22A. In a comparable experiment usingantibodies specific for HLA class 1, HLA-DR and CD40, the results shownin FIG. 22B were obtained.

Example 10 Cryoconservation of Dendritic Cells after Antigen Loading byMeans of RNA Transfection

Dendritic cells transfected with RNA can be frozen by the methodaccording to the invention in such a way that the properties of thedendritic cells are comparable with those of non-transfected dendriticcells. The DCs can be transfected with RNA in an immature stage, thenmatured, and then frozen as mature DCs (not shown). Preferably, DCswhich are already mature are transfected with RNA and cryoconserved. Theresults are summarized in FIGS. 25 to 27.

Thus, mature dendritic cells were washed twice with RPMI and once in awashing solution of the Optimix kit (EQUIBIO), Maidstone Kent, UK). DCswere brought to a final concentration of 40×10⁶ DCs/ml in Optimixmedium. Then, 0.1 ml of the cell suspension were mixed with 40 μg ofin-vitro transcribed EGFP RNA in a 1.5 ml reaction vessel. Afterincubation at room temperature for a maximum of 3 min, the cellsuspension was transferred into a 0.4 cm gap electroporation cuvette andpulsed at a voltage of 260 V and a capacitance of 150 μF with a GenePulser II (Biorad, Munich, Germany). Control DCs were pulsed without theaddition of RNA. The cells were frozen at a concentration of 10×10⁶DCs/ml in HSA (with 10% DMSO and 50% glucose (final concentration)) andstored for 4 hours. After thawing, the viability of the cells wasdetermined by trypan blue exclusion. The recovery rate of viable cellsis stated in FIG. 25 as a percentage of frozen DCs.

Mature DCs were electroporated with EGFP RNA and cryoconserved asdescribed above. After rethawing and a culturing period of 48 hours, thedendritic cells were co-cultured with allogenic CD4+ T cells (2×10⁵ perwell) under the conditions stated in FIG. 26. After 4 days, the cellswere pulsed with [³H]-thymidine for 16 hours, and the incorporatedradioactivity was determined. In FIG. 26, the average values fortriplicate measurements (with standard deviation) are shown. The valuesfor T cells alone or DCs alone were always less than 1000 per min.

The cryoconservation of RNA-electroporated DCs does not change thephenotypical DC marker. Thus, mature DCs were electroporated with orwithout EGFP RNA and cryoconserved as described above. After rethawingand a culturing time of 48 hours, the DCs were counterstained using themouse monoclonal antibodies stated in FIG. 27 and PE-conjugatedanti-mouse IG (Fab′)₂ fragments, followed by FACS analysis. The figuresin the right bottom portion of the square in FIG. 27 relate to theEGFP-positive DCs, and those in the top right portion relate to theEGFP/DC-marker double-positive DCs.

What is Claimed:

1. Dendritic cells frozen in freezing medium comprising autologousserum, 5-25% DMSO, and 5-10% glucose.
 2. The dendritic cells of claim 1which are mature dendritic cells.
 3. The dendritic cells of claim 1,wherein said cells are frozen in said freezing medium at a concentrationof from 5×10⁶ cells/ml to 20×10⁶ cells/ml.
 4. The dendritic cells ofclaim 1, wherein said dendritic cells are antigen-loaded.
 5. Thedendritic cells of claim 1, wherein said cells are frozen in saidfreezing medium at a concentration of 10×10⁶ cells/ml.
 6. The dendriticcells of claim 1, wherein said freezing medium contains from 10% to 20%DMSO.
 7. The dendritic cells of claim 4, wherein said dendritic cellsare loaded with a DNA or RNA molecule which codes for said antigen. 8.The dendritic cells of claim 1, wherein said dendritic cells are derivedfrom monocytes.
 9. The dendritic cells of claim 4, wherein saiddendritic cells are mature dendritic cells loaded with an RNA moleculethat codes for an antigen.
 10. The dendritic cells of claim 1, whereinsaid freezing medium contains about 10% DMSO and about 5% glucose. 11.The dendritic cells of claim 1, wherein said dendritic cells are maturedendritic cells loaded with an RNA molecule that codes for an antigenand wherein said freezing medium contains about 10% DMSO and about 5%glucose.